Alda-1 treatment and prevention of pulmonary disease

ABSTRACT

Method of treating or preventing allergic or pulmonary diseases characterized by endothelial dysfunction with Alda-1. Methods of treating or preventing endothelial dysfunction, and/or method of activating ALDH2 with Alda-1. Specifically, it was found that Alda-1 shields endothelial cells against oxidative stress via activation of ALDH2.

CROSS-REFERENCE TO RELATED APPLICATIONS

This nonprovisional application claims priority to U.S. Provisional Patent Application No. 62/634,539, entitled “ALDA-1 Shields Endothelial Cells Against Oxidative Stress Via Activation of ALDH2,” filed Feb. 23, 2018 by the same inventors, the entirety is incorporated herein by this reference.

FEDERAL FUNDING

This invention was made with government support HL105932 awarded by the National Institutes of Health. The Government has certain rights in the invention.

TECHNICAL FIELD

This invention relates, generally, to allergic and/or pulmonary diseases and the treatment of allergic and/or pulmonary diseases. More specifically, it relates to treating endothelial dysfunction and changes in vascular permeability.

BACKGROUND

Endothelial dysfunction and changes in vascular permeability are key events in many allergic diseases including edema and chronic obstructive pulmonary disease (COPD). Mitochondrial dysfunction causes elevation of reactive oxygen species (ROS). ALDH2 (aldehyde dehydrogenase 2) is known to be an effective combatant against mitochondrial dysfunction. Human carriers possessing an inactive ALDH2 variant have been found to be more susceptible to COPD and asthma. However, information pertaining to the ALDH2 interactome is limited.

Aldehyde dehydrogenases (ALDH) constitute a family of enzymes that play a critical role in detoxification of various cytotoxic xenogenic and biogenic aldehydes. ALDH is a key enzyme in fructose, acetaldehyde and oxalate metabolism and represents a major detoxification system for reactive carbonyls and aldehydes. There are at least 19 members/isozymes of the ALDH family, where the various isozymes may exhibit different substrate specificity and/or cellular localization relative to other members of the family.

Cytotoxic aldehydes derive from a variety of sources. For example, environmental (external) sources of aldehydes include those that result from ethanol consumption, from consumption of food sources, from ingestion of hazardous materials such as vinyl chloride, pesticides, herbicides, or from inhalation of hazardous materials such as those found in cigarette smoke, or industrial pollution. Aldehydes, that may be cytotoxic, can also be produced biologically (e.g., endogenously), e.g., as a result of oxidative stress such as occurs in ischemia, irradiation, or metabolism or bioconversion of cellular precursors such as neurotransmitters and drugs. Accumulation of cytotoxic levels of aldehydes, and/or defects in the ALDH enzyme, has been implicated in a variety of diseases and conditions, or in increased risk of disease development. The range of implicated diseases includes neurodegenerative diseases, aging, cancer, myocardial infarction, stroke, dermatitis, diabetes, cataracts, and liver diseases.

Accordingly, what is needed is a therapy targeted towards activation of ALDH2. However, in view of the art considered as a whole at the time the present invention was made, it was not obvious to those of ordinary skill in the field of this invention how the shortcomings of the prior art could be overcome.

While certain aspects of conventional technologies have been discussed to facilitate disclosure of the invention, Applicants in no way disclaim these technical aspects, and it is contemplated that the claimed invention may encompass one or more of the conventional technical aspects discussed herein.

The present invention may address one or more of the problems and deficiencies of the prior art discussed above. However, it is contemplated that the invention may prove useful in addressing other problems and deficiencies in a number of technical areas. Therefore, the claimed invention should not necessarily be construed as limited to addressing any of the particular problems or deficiencies discussed herein.

In this specification, where a document, act or item of knowledge is referred to or discussed, this reference or discussion is not an admission that the document, act or item of knowledge or any combination thereof was at the priority date, publicly available, known to the public, part of common general knowledge, or otherwise constitutes prior art under the applicable statutory provisions; or is known to be relevant to an attempt to solve any problem with which this specification is concerned.

SUMMARY

This summary is provided to introduce a selection of concepts in a simplified form.

These concepts are described in further detail in the detailed description of example embodiments of the disclosure below. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.

The present disclosure provides compounds that function as modulators of aldehyde dehydrogenase 2 (ALDH2) enzymatic activity, as well as compositions and formulations comprising the compounds. The present disclosure provides therapeutic methods involving administering a subject compound, or a subject pharmaceutical composition.

Embodiments disclosed herein include methods of preventing endothelial cell injury from hyperoxic injury, as well as related compounds. For example, in accordance with one embodiment, administering a therapeutically effective amount of Alda-1 and a pharmaceutically acceptable excipient, wherein the Alda-1 activates aldehyde dehydrogenase 2 (ALDH2), wherein the Alda-1 reduces damage to endothelial cells from oxidative stress.

In additional embodiments, the Alda-1 rescues mitochondrial membrane potential.

In further embodiments, the Alda-1 decreases cytochrome C release.

In additional embodiments, the Alda-1 suppresses mitochondrial reactive oxygen species production in human microvascular endothelial cells.

In additional embodiments, the mitochondria are preserved responsive to the administration of Alda-1.

The invention includes a method of treating pulmonary disease characterized by endothelial dysfunction, the method includes the steps of administering a therapeutically effective amount of Alda-1 to a subject suffering from the pulmonary disease characterized by endothelial dysfunction. A pharmaceutically acceptable excipient is then administered. The Alda-1 activates ALDH2 wherein the Alda-1 reduces damage to endothelial cells from oxidative stress.

BRIEF DESCRIPTION OF THE DRAWINGS

For a fuller understanding of the invention, reference should be made to the following detailed description, taken in connection with the accompanying drawings, in which:

FIG. 1 depicts MitoSOX images.

FIG. 2 depicts cells in normoxia, hyperoxia, and hyperoxia with Alda-1 at 24 hours.

FIG. 3 depicts cells in normoxia, hyperoxia, and hyperoxia with Alda-1 at 48 hours.

FIG. 4 depicts certain results of the present invention from normoxia, hyperoxia, and hyperoxia with Alda-1 on cytochrome C, beta actin, and beclin at 24 hours and 48 hours.

FIG. 5 shows a graph that represent the whole cell protein lysate for Human microvascular endothelial cells under different conditions subjected to ELISA to evaluate the levels of 4HNE content during the Normoxia (NO), hyperoxia (HO), hyperoxia+Alda1 (HO+Alda-1) (20 μM) for 48 hours. The results are shown in mean±SEM.

FIG. 6A shows the western blot analysis of the whole cell lysate for human microvascular endothelial cells under different conditions (n=3) subjected evaluate the levels of cytochrome C during normoxia, hyperoxia and hyperoxia with Alda-1 (20 μM) for 48 hours. Equal amounts of protein loaded per lane (20 μg).

FIG. 6B shows expression of Cytochrome C was normalized to Beta actin and presented in arbitrary units. The results are shown in mean±SEM.

FIG. 7A shows a graph that depicts the whole cell lysate for human microvascular endothelial cells under different conditions (n=3) subjected to Immuno blot to evaluate the levels of ALDH2 during normoxia, hyperoxia and hyperoxia with Alda-1 (20 μM) for 48 hours normalized against Beta actin. The values indicate standard error mean (*P<0.05). Equal amounts of protein loaded per lane (20 μg).

FIG. 7B shows expression of ALDH2 was normalized to Beta actin and presented in arbitrary units. The results are shown in mean±SEM.

FIG. 8A shows a graph that depicts the whole cell lysate for human microvascular endothelial cells under different conditions (n=3) subjected to Immuno blot to evaluate the levels of Bax during normoxia, hyperoxia and hyperoxia with Alda-1 (20 μM) for 48 hours normalized against Beta actin. The values indicate standard error mean (*P<0.05). Equal amounts of protein loaded per lane (20 μg).

FIG. 8B shows expression of Bax was normalized to Beta actin and presented in arbitrary units. The results are shown in mean±SEM.

FIG. 9A shows a graph that shows the human microvascular endothelial cells under different conditions subjected to the JC1 staining to evaluate the mitochondrial membrane potential during normoxia, hyperoxia and hyperoxia with Alda-1 (20 μM) for 48 hours. The values indicate standard error mean (*P<0.05).

FIG. 9B depicts Jc1 images were quantified using imageJ software and an intensity was expressed in arbitrary units. The results are shown in mean±SEM (Magnification=200×).

FIG. 10 shows a graph that shows the mitochondrial lysate for the human microvascular endothelial cells under different conditions (n=3) subjected to enzymatic assay to evaluate the ALDH2 activity at 340 nm during normoxia, hyperoxia and hyperoxia with Alda-1 (20 μM) for 48 hours.

DETAILED DESCRIPTION

In the following detailed description, reference is made to the accompanying drawings, which form a part thereof, and within which are shown by way of illustration specific embodiments by which the invention may be practiced. It is to be understood that other embodiments may be used, and structural changes may be made without departing from the scope of the present application. These embodiments are described in sufficient detail to enable those of ordinary skill in the art to practice the present disclosure, and it is to be understood that other embodiments may be utilized, and that structural, logical, and electrical changes may be made within the scope of the disclosure.

From the following descriptions, it should be understood that components of the embodiments as generally described and illustrated in the figures herein could be arranged and designed in a wide variety of different configurations. Thus, the following more detailed description of various embodiments, as represented in the figures, is not intended to limit the scope of the disclosure but is merely representative of various embodiments. While the various aspects of the embodiments are presented in drawings, the drawings are not necessarily drawn to scale unless specifically indicated.

The following description provides specific details, such as material types, compositions, material thicknesses, and processing conditions in order to provide a thorough description of embodiments of the disclosure. However, a person of ordinary skill in the art will understand that the embodiments of the disclosure may be practiced without employing these specific details. Indeed, the embodiments of the disclosure may be practiced in conjunction with conventional techniques employed in the industry. Only those process acts and structures necessary to understand the embodiments of the disclosure are described in detail below. A person of ordinary skill in the art will understand that some process components are inherently disclosed herein and that adding various conventional process components and acts would be in accord with the disclosure. In this description, specific implementations are shown and described only as examples and should not be construed as the only way to implement the present disclosure unless specified otherwise herein.

Illustrations presented herein are not meant to be actual views of any particular material, component, or system, but are merely idealized representations that are employed to describe embodiments of the disclosure. Referring in general to the following description and accompanying drawings, various embodiments of the present disclosure are illustrated to show its structure and method of operation. Common elements of the illustrated embodiments may be designated with similar reference numerals. It should be understood that the figures presented are not meant to be illustrative of actual views of any particular portion of the actual structure or method but are merely idealized representations employed to more clearly and fully depict the present invention defined by the claims below.

It should be understood that any reference to an element herein using a designation such as “first,” “second,” and so forth does not limit the quantity or order of those elements, unless such limitation is explicitly stated. Rather, these designations may be used herein as a convenient method of distinguishing between two or more elements or instances of an element. Thus, a reference to first and second elements does not mean that only two elements may be employed there or that the first element must precede the second element in some manner. Also, unless stated otherwise a set of elements may comprise one or more elements.

Any headings used herein should not be considered to limit the scope of embodiments of the invention as defined by the claims below and their legal equivalents. Concepts described in any specific heading are generally applicable in other sections throughout the entire specification.

As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the content clearly dictates otherwise. As used in this specification and the appended claims, the term “or” is generally employed in its sense including “and/or” unless the context clearly dictates otherwise.

In an embodiment, the current invention includes an activator of ALDH2, specifically

Alda-1, that amplifies ALDH2 activity and attenuates endothelial dysfunction in pulmonary diseases. Alda-1 also known as N-(1,3-Benzodioxol-5-ylmethyl)-2,6-dichlorobenzamid has the empirical formula C₁₅H₁₁Cl₂NO₃ with molecular weight 324.16. Alda-1 has structure:

As used herein, the term “aldehyde dehydrogenase” or “ALDH” refers to an enzyme that oxidizes an aldehyde (e.g., a xenogenic aldehyde, a biogenic aldehyde, or an aldehyde produced from a compound that is ingested, inhaled, or absorbed) to its corresponding acid in an NAD+-dependent or an NADP+-dependent reaction. For example, ALDH oxidizes aldehydes derived from the breakdown of compounds, e.g., toxic compounds that are ingested, that are absorbed, that are inhaled, that are produced as a result of oxidative stress, or that are produced during normal metabolism, e.g., conversion of retinaldehyde to retinoic acid. An example of a biogenic aldehyde is acetaldehyde produced as a product of alcohol dehydrogenase activity on ingested ethanol. An aldehyde dehydrogenase can also exhibit esterase activity and/or reductase activity.

The term “ALDH” encompasses ALDH found in the cytosol, in the mitochondria, microsome, or other cellular compartment. The term “ALDH” encompasses ALDH found primarily in one or a few tissues, e.g., cornea, saliva, liver, etc., or in stem cells and embryos. The term “ALDH” encompasses any of the known ALDH isozymes, including ALDH1, ALDH2, ALDH3, ALDH4, ALDHS, etc.

As used herein, the term “mitochondrial aldehyde dehydrogenase-2” or “ALDH2” refers to an enzyme that oxidizes an aldehyde (e.g., a xenogenic aldehyde, a biogenic aldehyde, or an aldehyde produced from a compound that is ingested, inhaled, or absorbed) to its corresponding acid in an NAD+-dependent reaction. For example, ALDH2 oxidizes aldehydes derived from the breakdown of compounds, e.g., toxic compounds that are ingested, that are absorbed, that are inhaled, or that are produced during normal metabolism. Mitochondrial ALDH2 is naturally found in mitochondria.

The term “ALDH2” encompasses ALDH2 from various species. Amino acid sequences of ALDH2 from various species are publicly available. For example, a human ALDH2 amino acid sequence is found under GenBank Accession Nos. AAH02967 and NP-000681; a mouse ALDH2 amino acid sequence is found under GenBank Accession No. NP-033786; and a rat ALDH2 amino acid sequence is found under GenBank Accession No. NP-115792. The term “ALDH2” encompasses an aldehyde dehydrogenase that exhibits substrate specificity, e.g., that preferentially oxidizes aliphatic aldehydes.

The term “ALDH2” as used herein also encompasses fragments, fusion proteins, and variants (e.g., variants having one or more amino acid substitutions, addition, deletions, and/or insertions) that retain ALDH2 enzymatic activity. Specific enzymatically active ALDH2 variants, fragments, fusion proteins, and the like can be verified by adapting the methods described herein. “ALDH2” includes an enzyme that converts acetaldehyde into acetic acid, e.g., where the acetaldehyde is formed in vivo by the action of alcohol dehydrogenase on ingested ethanol.

As used herein, “about” means approximately or nearly and in the context of a numerical value or range set forth means±15% of the numerical. In an embodiment, the term “about” can include traditional rounding according to significant figures of the numerical value. In addition, the phrase “about ‘x’ to ‘y’” includes “about ‘x’ to about ‘y’”.

It should be noted that ratios, concentrations, amounts, and other numerical data may be expressed herein in a range format. It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a concentration range of “about 0.1% to about 5%” should be interpreted to include not only the explicitly recited concentration of about 0.1 wt % to about 5 wt %, but also include individual concentrations (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.5%, 1.1%, 2.2%, 3.3%, and 4.4%) within the indicated range.

As used herein, the term “subject,” “patient,” or “organism” includes humans and mammals (e.g., mice, rats, pigs, cats, dogs, and horses). Typical hosts to which an agent(s) of the present disclosure may be administered will be mammals, particularly primates, especially humans. For veterinary applications, a wide variety of subjects will be suitable, e.g., livestock such as cattle, sheep, goats, cows, swine, and the like; poultry such as chickens, ducks, geese, turkeys, and the like; and domesticated animals particularly pets such as dogs and cats. For diagnostic or research applications, a wide variety of mammals will be suitable subjects, including rodents (e.g., mice, rats, hamsters), rabbits, primates, and swine such as inbred pigs and the like.

The phrases “connected to” and “coupled to” refer to any form of interaction between two or more entities, including mechanical, electrical, magnetic, electromagnetic, fluid, and thermal interaction. Two components may be connected or coupled to each other even though they are not in direct contact with each other. For example, two components may be coupled to each other through an intermediate component.

As used herein, “treat”, “treatment”, “treating”, and the like refer to acting upon a condition (e.g., endothelial dysfunction) with an agent (e.g., Alda-1) to affect the condition by improving or altering it. The improvement or alteration may include an improvement in symptoms or an alteration in the physiologic pathways associated with the condition. The aforementioned terms cover one or more treatments of a condition in a patient (e.g., a mammal, typically a human or non-human animal of veterinary interest), and includes: (a) reducing the risk of occurrence of the condition in a subject determined to be predisposed to the condition but not yet diagnosed, (b) impeding the development of the condition, and/or (c) relieving the condition, e.g., causing regression of the condition and/or relieving one or more condition symptoms.

As used herein, the terms “prophylactically treat” or “prophylactically treating” refers completely or partially preventing (e.g., about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, or about 99% or more) a condition or symptom thereof and/or may be therapeutic in terms of a partial or complete cure or alleviation for a condition and/or adverse effect attributable to the condition.

A “pharmaceutically acceptable excipient,” “pharmaceutically acceptable diluent,” “pharmaceutically acceptable carrier,” or “pharmaceutically acceptable adjuvant” means an excipient, diluent, carrier, and/or adjuvant that are useful in preparing a pharmaceutical composition that are generally safe, non-toxic and neither biologically nor otherwise undesirable, and include an excipient, diluent, carrier, and adjuvant that are acceptable for veterinary use and/or human pharmaceutical use. “A pharmaceutically acceptable excipient, diluent, carrier and/or adjuvant” as used in the specification and claims includes one or more such excipients, diluents, carriers, and adjuvants.

The term “therapeutically effective amount” as used herein describes concentrations or amounts of components such as agents which are effective for producing an intended result, including attenuation of endothelial dysfunction. Compositions according to the present invention may be used to effect a favorable change in endothelial dysfunction, whether that change is an improvement, relieving to some extent one or more of the symptoms of the condition being treated, and/or that amount that will prevent, to some extent, one or more of the symptoms of the condition that the host being treated has or is at risk of developing, or a complete cure of the disease or condition treated.

The term “isolated compound” means a compound which has been substantially separated from, or enriched relative to, other compounds with which it occurs in nature. Isolated compounds are at least about 80%, at least about 90% pure, at least about 98% pure, or at least about 99% pure, by weight. The present disclosure is meant to comprehend diastereomers as well as their racemic and resolved, enantiomerically pure forms and pharmaceutically acceptable salts thereof.

The term “administration” or “administering” is used throughout the specification to describe the process by which a composition comprising Alda-1 as an active agent, are delivered to a patient or individual for therapeutic purposes. The composition of the subject invention and methodology in use thereof can be administered a number of ways including, but not limited to, parenteral (such term referring to intravenous and intra-arterial as well as other appropriate parenteral routes), subcutaneous, peritoneal, inhalation, vaginal, rectal, nasal, or instillation into body compartments.

Administration will often depend upon the amount of compound administered, the number of doses, and duration of treatment. In an embodiment, multiple doses of the agent are administered. The frequency of administration of the agent can vary depending on any of a variety of factors, such as attenuation of endothelial dysfunction and the like. The duration of administration of the agent, e.g., the period of time over which the agent is administered, can vary, depending on any of a variety of factors, including patient response, etc.

The amount of the agent contacted (e.g., administered) can vary according to factors such as the degree of susceptibility of the individual, the age, sex, and weight of the individual, idiosyncratic responses of the individual, the dosimetry, and the like. Detectably effective amounts of the agent of the present disclosure can also vary according to instrument and film-related factors. Optimization of such factors is well within the level of skill in the art, unless otherwise noted.

“In combination with,” or “co-administration,” as used herein, in the context of administering a first compound and at least a second compound, refers to uses where, for example, the first compound is administered during the entire course of administration of the second compound; where the first compound is administered for a period of time that is overlapping with the administration of the second compound, e.g. where administration of the first compound begins before the administration of the second compound and the administration of the first compound ends before the administration of the second compound ends; where the administration of the second compound begins before the administration of the first compound and the administration of the second compound ends before the administration of the first compound ends; where the administration of the first compound begins before administration of the second compound begins and the administration of the second compound ends before the administration of the first compound ends; where the administration of the second compound begins before administration of the first compound begins and the administration of the first compound ends before the administration of the second compound ends. As such, “in combination” can also refer to regimen involving administration of two or more compounds. “In combination with” as used herein also refers to administration of two or more compounds which may be administered in the same or different formulations, by the same of different routes, and in the same or different dosage form type.

The term “unit dosage form,” as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of compounds of the present disclosure calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for the novel unit dosage forms of the present disclosure depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.

The term “physiological conditions” is meant to encompass those conditions compatible with living cells, e.g., predominantly aqueous conditions of a temperature, pH, salinity, etc. that are compatible with living cells.

“Pro-drugs” means any compound that releases an active parent drug according to one or more of the generic formulas shown below in vivo when such prodrug is administered to a mammalian subject. Prodrugs of a compound of one or more of the generic formulas shown below are prepared by modifying functional groups present in the compound of the generic formula in such a way that the modifications may be cleaved in vivo to release the parent compound. Prodrugs include compounds of one or more of the generic formulas shown below wherein a hydroxy, amino, or sulfhydryl group in one or more of the generic formulas shown below is bonded to any group that may be cleaved in vivo to regenerate the free hydroxyl, amino, or sulfhydryl group, respectively. Examples of prodrugs include, but are not limited to esters (e.g., acetate, formate, and benzoate derivatives), carbamates (e.g., N,N-dimethylaminocarbonyl) of hydroxy functional groups in compounds of one or more of the generic formulas shown below, and the like.

The term “organic group” and “organic radical” as used herein means any carbon-containing group, including hydrocarbon groups that are classified as an aliphatic group, cyclic group, aromatic group, functionalized derivatives thereof and/or various combinations thereof. The term “aliphatic group” means a saturated or unsaturated linear or branched hydrocarbon group and encompasses alkyl, alkenyl, and alkynyl groups, for example. The term “alkyl group” means a substituted or unsubstituted, saturated linear or branched hydrocarbon group or chain (e.g., C1 to C8) including, for example, methyl, ethyl, isopropyl, tert-butyl, heptyl, iso-propyl, n-octyl, dodecyl, octadecyl, amyl, 2-ethylhexyl, and the like. Suitable substituents include carboxy, protected carboxy, amino, protected amino, halo, hydroxy, protected hydroxy, nitro, cyano, monosubstituted amino, protected monosubstituted amino, disubstituted amino, C1 to C7 alkoxy, C1 to C7 acyl, C1 to C7 acyloxy, and the like. The term “substituted alkyl” means the above defined alkyl group substituted from one to three times by a hydroxy, protected hydroxy, amino, protected amino, cyano, halo, trifloromethyl, mono-substituted amino, di-substituted amino, lower alkoxy, lower alkylthio, carboxy, protected carboxy, or a carboxy, amino, and/or hydroxy salt. As used in conjunction with the substituents for the heteroaryl rings, the terms “substituted (cycloalkyl)alkyl” and “substituted cycloalkyl” are as defined below substituted with the same groups as listed for a “substituted alkyl” group. The term “alkenyl group” means an unsaturated, linear or branched hydrocarbon group with one or more carbon-carbon double bonds, such as a vinyl group. The term “alkynyl group” means an unsaturated, linear or branched hydrocarbon group with one or more carbon-carbon triple bonds.

The term “cyclic group” means a closed ring hydrocarbon group that is classified as an alicyclic group, aromatic group, or heterocyclic group. The term “alicyclic group” means a cyclic hydrocarbon group having properties resembling those of aliphatic groups. The term “aromatic group” or “aryl group” means a mono- or polycyclic aromatic hydrocarbon group, and may include one or more heteroatoms, and which are further defined below. The term “heterocyclic group” means a closed ring hydrocarbon in which one or more of the atoms in the ring are an element other than carbon (e.g., nitrogen, oxygen, sulfur, etc.), and are further defined below.

“Organic groups” may be functionalized or otherwise comprise additional functionalities associated with the organic group, such as carboxyl, amino, hydroxyl, and the like, which may be protected or unprotected. For example, the phrase “alkyl group” is intended to include not only pure open chain saturated hydrocarbon alkyl substituents, such as methyl, ethyl, propyl, t-butyl, and the like, but also alkyl substituents bearing further substituents known in the art, such as hydroxy, alkoxy, alkylsulfonyl, halogen atoms, cyano, nitro, amino, carboxyl, etc. Thus, “alkyl group” includes ethers, esters, haloalkyls, nitroalkyls, carboxyalkyls, hydroxyalkyls, sulfoalkyls, etc.

The terms “halo” and “halogen” refer to the fluoro, chloro, bromo or iodo groups. There can be one or more halogen, which are the same or different. Halogens of particular interest include chloro and bromo groups.

The term “haloalkyl” refers to an alkyl group as defined above that is substituted by one or more halogen atoms. The halogen atoms may be the same or different. The term “dihaloalkyl” refers to an alkyl group as described above that is substituted by two halo groups, which may be the same or different. The term “trihaloalkyl” refers to an alkyl group as describe above that is substituted by three halo groups, which may be the same or different. The term “perhaloalkyl” refers to a haloalkyl group as defined above wherein each hydrogen atom in the alkyl group has been replaced by a halogen atom. The term “perfluoroalkyl” refers to a haloalkyl group as defined above wherein each hydrogen atom in the alkyl group has been replaced by a fluoro group.

The term “cycloalkyl” means a mono-, bi-, or tricyclic saturated ring that is fully saturated or partially unsaturated. Examples of such a group included cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, cyclooctyl, cis- or trans decalin, bicyclo[2.2.1]hept-2-ene, cyclohex-1-enyl, cyclopent-1-enyl, 1,4-cyclooctadienyl, and the like.

The term “(cycloalkyl)alkyl” means the above-defined alkyl group substituted for one of the above cycloalkyl rings. Examples of such a group include (cyclohexyl)methyl, 3-(cyclopropyl)-n-propyl, 5-(cyclopentyl)hexyl, 6-(adamantyl)hexyl, and the like.

The term “substituted phenyl” specifies a phenyl group substituted with one or more moieties, and in some instances one, two, or three moieties, chosen from the groups consisting of halogen, hydroxy, protected hydroxy, cyano, nitro, trifluoromethyl, C1 to C7 alkyl, C1 to C7 alkoxy, C1 to C7 acyl, C1 to C7 acyloxy, carboxy, oxycarboxy, protected carboxy, carboxymethyl, protected carboxymethyl, hydroxymethyl, protected hydroxymethyl, amino, protected amino, (monosubstituted)amino, protected (monosubstituted)amino, (disubstituted)amino, carboxamide, protected carboxamide, N-(C1 to C6 alkyl)carboxamide, protected N-(C1 to C6 alkyl)carboxamide, N,N-di(C1 to C6 alkyl)carboxamide, trifluoromethyl, N-((C1 to C6 alkyl)sulfonyl)amino, N-(phenylsulfonyl)amino or phenyl, substituted or unsubstituted, such that, for example, a biphenyl or naphthyl group results.

Examples of the term “substituted phenyl” includes a mono- or di(halo)phenyl group such as 2, 3 or 4-chlorophenyl, 2,6-dichlorophenyl, 2,5-dichlorophenyl, 3,4-dichlorophenyl, 2,3 or 4-bromophenyl, 3,4-dibromophenyl, 3-chloro-4-fluorophenyl, 2,3 or 4-fluorophenyl and the like; a mono or di(hydroxy)phenyl group such as 2,3, or 4-hydroxyphenyl, 2,4-dihydroxyphenyl, the protected-hydroxy derivatives thereof and the like; a nitrophenyl group such as 2,3, or 4-nitrophenyl; a cyanophenyl group, for example, 2,3 or 4-cyanophenyl; a mono- or di(alkyl)phenyl group such as 2,3, or 4-methylphenyl, 2,4-dimethylphenyl, 2,3 or 4-(iso-propyl)phenyl, 2,3, or 4-ethylphenyl, 2,3 or 4-(n-propyl)phenyl and the like; a mono or di(alkoxy)phenyl group, for example, 2,6-dimethoxyphenyl, 2,3 or 4-(isopropoxy)phenyl, 2,3 or 4-(t-butoxy)phenyl, 3-ethoxy-4-methoxyphenyl and the like; 2,3 or 4-trifluoromethylphenyl; a mono- or dicarboxyphenyl or (protected carboxy)phenyl group such as 2,3 or 4-carboxyphenyl or 2,4-di(protected carboxy)phenyl; a mono- or di(hydroxymethyl)phenyl or (protected hydroxymethyl)phenyl such as 2,3 or 4-(protected hydroxymethyl)phenyl or 3,4-di(hydroxymethyl)phenyl; a mono- or di(aminomethyl)phenyl or (protected aminomethyl)phenyl such as 2,3 or 4-(aminomethyl)phenyl or 2,4-(protected aminomethyl)phenyl; or a mono- or di(N-(methylsulfonylamino))phenyl such as 2,3 or 4-(N-(methylsulfonylamino))phenyl. Also, the term “substituted phenyl” represents disubstituted phenyl groups wherein the substituents are different, for example, 3-methyl-4-hydroxyphenyl, 3-chloro-4-hydroxyphenyl, 2-methoxy-4-bromophenyl, 4-ethyl-2-hydroxyphenyl, 3-hydroxy-4-nitrophenyl, 2-hydroxy-4-chlorophenyl, and the like.

The term “(substituted phenyl)alkyl” means one of the above substituted phenyl groups attached to one of the above-described alkyl groups. Examples of include such groups as 2-phenyl-1-chloroethyl, 2-(4′-methoxyphenyl)ethyl, 4-(2′,6′-dihydroxy phenyl)n-hexyl, 2-(5′-cyano-3′-methoxyphenyl)n-pentyl, 3-(2′,6′-dimethylphenyl)n-propyl, 4-chloro-3-aminobenzyl, 6-(4′-methoxyphenyl)-3-carboxy(n-hexyl), 5-(4′-aminomethylphenyl) -3-(aminomethyl)n-pentyl, 5-phenyl-3-oxo-n-pent-1-yl, (4-hydroxynapth-2-yl)methyl and the like.

As noted above, the term “aromatic” or “aryl” refers to six membered carbocyclic rings. Also as noted above, the term “heteroaryl” denotes optionally substituted five-membered or six-membered rings that have 1 to 4 heteroatoms, such as oxygen, sulfur and/or nitrogen atoms, in particular nitrogen, either alone or in conjunction with sulfur or oxygen ring atoms.

Furthermore, the above optionally substituted five-membered or six-membered rings can optionally be fused to an aromatic 5-membered or 6-membered ring system. For example, the rings can be optionally fused to an aromatic 5-membered or 6-membered ring system such as a pyridine or a triazole system, and preferably to a benzene ring.

The following ring systems are examples of the heterocyclic (whether substituted or unsubstituted) radicals denoted by the term “heteroaryl”: thienyl, furyl, pyrrolyl, pyrrolidinyl, imidazolyl, isoxazolyl, triazolyl, thiadiazolyl, oxadiazolyl, tetrazolyl, thiatriazolyl, oxatriazolyl, pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, oxazinyl, triazinyl, thiadiazinyl tetrazolo, 1,5-[b]pyridazinyl and purinyl, as well as benzo-fused derivatives, for example, benzoxazolyl, benzthiazolyl, benzimidazolyl and indolyl.

Substituents for the above optionally substituted heteroaryl rings are from one to three halo, trihalomethyl, amino, protected amino, amino salts, mono-substituted amino, di-substituted amino, carboxy, protected carboxy, carboxylate salts, hydroxy, protected hydroxy, salts of a hydroxy group, lower alkoxy, lower alkylthio, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, (cycloalkyl)alkyl, substituted (cycloalkyl)alkyl, phenyl, substituted phenyl, phenylalkyl, and (substituted phenyl)alkyl. Substituents for the heteroaryl group are as heretofore defined, or in the case of trihalomethyl, can be trifluoromethyl, trichloromethyl, tribromomethyl, or triiodomethyl. As used in conjunction with the above substituents for heteroaryl rings, “lower alkoxy” means a C1 to C4 alkoxy group, similarly, “lower alkylthio” means a C1 to C4 alkylthio group.

The term “(monosubstituted)amino” refers to an amino group with one substituent chosen from the group consisting of phenyl, substituted phenyl, alkyl, substituted alkyl, C1 to C4 acyl, C2 to C7 alkenyl, C2 to C7 substituted alkenyl, C2 to C7 alkynyl, C7 to C16 alkylaryl, C7 to C16 substituted alkylaryl and heteroaryl group. The (monosubstituted) amino can additionally have an amino-protecting group as encompassed by the term “protected (monosubstituted)amino.” The term “(disubstituted)amino” refers to amino groups with two substituents chosen from the group consisting of phenyl, substituted phenyl, alkyl, substituted alkyl, C1 to C7 acyl, C2 to C7 alkenyl, C2 to C7 alkynyl, C7 to C16 alkylaryl, C7 to C16 substituted alkylaryl and heteroaryl. The two substituents can be the same or different.

The term “heteroaryl(alkyl)” denotes an alkyl group as defined above, substituted at any position by a heteroaryl group, as above defined.

“Optional” or “optionally” means that the subsequently described event, circumstance, feature, or element may, but need not, occur, and that the description includes instances where the event or circumstance occurs and instances in which it does not. For example, “heterocyclo group optionally mono- or di-substituted with an alkyl group” means that the alkyl may, but need not, be present, and the description includes situations where the heterocyclo group is mono- or disubstituted with an alkyl group and situations where the heterocyclo group is not substituted with the alkyl group.

Compounds that have the same molecular formula but differ in the nature or sequence of bonding of their atoms or the arrangement of their atoms in space are termed “isomers.” Isomers that differ in the arrangement of their atoms in space are termed “stereoisomers.” Stereoisomers that are not mirror images of one another are termed “diastereomers” and those that are non-superimposable mirror images of each other are termed “enantiomers.” When a compound has an asymmetric center, for example, it is bonded to four different groups, a pair of enantiomers is possible. An enantiomer can be characterized by the absolute configuration of its asymmetric center and is described by the R- and S-sequencing rules of Cahn and Prelog, or by the manner in which the molecule rotates the plane of polarized light and designated as dextrorotatory or levorotatory (i.e., as (+) or (−)-isomers respectively). A chiral compound can exist as either individual enantiomer or as a mixture thereof. A mixture containing equal proportions of the enantiomers is called a “racemic mixture.”

Referring in general to the following description and accompanying drawings, various embodiments of the present disclosure are illustrated to show its structure and method of operation. Common elements of the illustrated embodiments may be designated with similar reference numerals. Accordingly, the relevant descriptions of such features apply equally to the features and related components among all the drawings. Any suitable combination of the features, and variations of the same, described with components illustrated in FIG. 1, can be employed with the components of FIG. 2, and vice versa. This pattern of disclosure applies equally to further embodiments depicted in subsequent figures and described hereinafter. It should be understood that the figures presented are not meant to be illustrative of actual views of any particular portion of the actual structure or method but are merely idealized representations employed to more clearly and fully depict the present invention defined by the claims below.

All referenced publications are incorporated herein by reference in their entirety.

Furthermore, where a definition or use of a term in a reference, which is incorporated by reference herein, is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

Example 1

It is an object of the current invention to evaluate the effects of Alda-1 on lung endothelial cells during hyperoxic injury.

Materials and Methods. Cells obtained were human microvascular endothelial cells (HMVECs). Cell culture medium was EBM-2 containing 10% FBS plus antibiotics. Assays were MitoSOX, JC1, Histology, Western blot, and ALDH2 activity. Antibodies used were mouse anti-ALDH2 antibody, Cytochrome C, and Beclin (cell signaling).

HMVECs were seeded and, after reaching confluence, treated with DMSO and Alda-1. Cells were then exposed to normoxia or hyperoxia at different time points. After 24, 48, and 72 hours, cells were collected for mitochondrial analyses.

Results. Cells treated with Alda-1 displayed elevated ALDH2 activity during both normoxia and hyperoxia. It was also observed that Alda-1 rescued mitochondrial membrane potential, decreased Cytochrome C release, and suppressed mitochondrial ROS production in HMVECs. See FIGS. 1-4.

FIG. 1 depicts MitoSOX images. FIG. 2 depicts cells at 24 hours which are exposed to normoxia, hyperoxia, and cells treated with Alda-1. FIG. 3 depicts cells at 48 hours which are exposed to normoxia, hyperoxia, and cells treated with Alda-1. FIG. 4 depicts certain results of the present invention. FIG. 4 depicts normoxia, hyperoxia, and the results of Alda-1 on hyperoxia cells on cytochrome C, Beclin, and Beta actin at 24 hours and 48 hours.

Conclusion. The findings suggest that Alda-1-mediated modulation of ALDH2 is an effective target for remediation of endothelial dysfunction through preservation of mitochondria. These results reveal a promising therapeutic approach to treating allergic diseases where endothelial dysfunction is a characteristic event.

Example 2—prophetic

It is an object of the current invention to evaluate the effects of Alda-1 on lung endothelial cells in vivo during hyperoxic injury.

Materials and Methods. Lung endothelial cells will be treated with Alda-1 and a pharmaceutically acceptable excipient. Cells will then be exposed to normoxia or hyperoxia at different time points. After 24, 48, and 72 hours, cells will be collected for mitochondrial analyses.

Results. Cells treated with Alda-1 will display elevated ALDH2 activity during both normoxia and hyperoxia. It will also be observed that Alda-1 rescued mitochondrial membrane potential, decreased Cytochrome C release, and suppressed mitochondrial ROS production in lung endothelial cells.

Example 3

Acute lung injury is caused by lengthened exposure to hyperoxia leads to oxidative stress by crucially impairing the pulmonary function. Mitochondrial dysfunction is a main event in hyperoxic acute lung injury It is well known fact that hyperoxia causes cellular damage and death by impairing mitochondrial Aldehyde dehydrogenase 2 (1) by inducing oxidative stress. Examining the capacity of Alda-1 (benzodioxyl dichlororobezamide) to shield the oxidative stress in HMVEC (2). HMVEC were pretreated with 20 μM of Alda-1 prior to exposure of hyperoxia shown a diminish in accumulation of toxic compound 4HNE, significant decrease in oxidative stress and apoptotic activity. Furthermore, Alda-1 pretreated samples in hyperoxia shown improved Aldh2 activity and ALDH2 expression and significantly enriched mitochondrial membrane potential. Therefore, activation of ALDH2 may inhibit the endothelial dysfunction caused by hyperoxic exposure.

Introduction. Acute lung injury (ALI) (3) is a serious clinical complication of respiratory fiasco affecting 200,000 people in US annually (4) and the cases are increasing yearly. The ALI furnish the death of 70,000 people alone in USA (5). The most similar type of ALI is acute respiratory distress syndrome (ARDS) and hyperoxia is an essential part of treatment (6). The routine treatment involves continuously exposure to hyperoxia and after effects are injurious to the patient (7). The prolonged exposure causes hyperoxic acute lung injury (HALI) eventually lead to death as shown many animal models. The oxidative stress, ROS and apoptosis is caused by hyperoxic exposure leads to dysfunction of endothelial cells (7-9).

The reason to focus on HALI because it has negligible amount of fibrosis in differentiation when compared to other lung injury models and hyperoxia helps in mimicking ALI (10) (5). The hyperoxia exposure generates oxidative stress, specially promote 4HNE a toxic compound causing harmful effects to mitochondria and impairing cell transduction has been proven by our past reports (9, 11). The 4HNE is a lipid peroxidation product, an ominous reactive aldehyde acts as biomarker for oxidative stress (8). The oxidative stress generated 4HNE deploy harmful impact on variety of tissues caridiac, neural, epithelial cells, endothelial cells (Chen, 2008; Galam, 2015; Neely, 1999; Rahman, 2002; Solito, 2013).

There are various therapies to metabolize the oxidative stress, but in lungs the Aldh2 acts as a therapeutic agent and also a substrate of 4HNE (9, 12-15). The other remedy for the acute lung injury to diminish 4HNE and ROS by modification of the molecule's membrane receptor px27 (16) and apoptosis signaling kinase (17). The ALDH2 activator Alda-1 has been studied and administration is rarely utilized in experiments (18). The use of Alda-1 successfully able to enhance the function of ALDH2 by reducing lung ischemia in epithelial cells, cerebral ischemia, cardiac ischemia, human umbilical endothelial cells by reducing the toxic 4HNE effectively (2, 12, 15, 19)

Until now there was no study conducted on human microvascular endothelial cell (HMVEC) in hyperoxia with an Aldh2 activator. The HMVEC play an important role in vascular function and homeostasis in the lungs, also involved in remodeling of vascular wall, angiogenesis, coagulation, blood vessel formation, acts as an interface for blood circulation (20). The HMVEC are vulnerable to the oxidative stress and hemodynamics alteration (7). Hyperoxia caused oxidative stress is a crucial replica that induce the harmful outcome of ALI by obstructing barriers of endothelial and epithelial cells (Galam, 2016). The long term exposure to hyperoxia causes buildup of ROS, affects cell proliferation, cell toxicity and cell viability (7). Therefore, Hyperoxia yield a foundation to probe the pathogenesis of cellular damage and pulmonary disease.

The effect of Alda-1 was measured on the hyperoxia to evaluate accumulation of 4HNE in hyperoxia. Due to hyperoxia the oxidative stress will be increased and leads to apoptosis, then we evaluated the Alda-1 effect on expression of cytochrome C and Bax in hyperoxia. Increase of 4HNE in HMVEC and prediction was made that mitochondrial function will be affected so effect of Alda-1 was on mitochondria was evaluated for mitochondrial membrane potential. Further, effect of Alda-1 on hyperoxia was measured to evaluate the expression and activity of ALDH2. Our results specified that effect of Alda-1 on hyperoxia reduced accumulation of 4HNE, oxidative stress, apoptosis and enhanced mitochondrial membrane potential and activity of ALDH2. Particularly the use of Alda-1 in hyperoxia that act as agonist to ALDH2 suppress the damages caused by the oxidative stress (12, 15, 18). This treatment provides a perception into auspicious replacement remedy in endothelial dysfunction caused by hyperoxic lung injury.

Materials and Methods.

Cell culture

HMVEC's were preserved in EGM-2 MV media (Lonza, Wakersville, Md.) a supplied with FBS (25 ml), Hydrocortisone (0.2 ml), hFGF-B (2.0 ml), VEGF (0.5 ml), R3 IGF-1 (0.5 ml), Ascorbic acid (0.5 ml), hEGF (0.5 ml), GA-1000 (0.5 ml) at 37 degrees Celsius in a 5% carbon dioxide humidified incubator to maintain a sufficient cell growth. The cultured celled validated for the confluence (around 70%) and exposed to hyperoxia for 48 hours with and without alda-1.

Oxidative stress assay

The samples of whole cell lysate (50 μg) were seeded in each well. The oxidative stress was measured by oxiselect 4HNE adduct competitive ELISA kit (Cell Biolabs), according to instructions of kit.

Western Blot

The HMVEC-L cells were cultured and deemed for the confluence (70%) and exposed to hyperoxia with or without alda-1 for 48 hours. The concentration was determined by the proOX p100 sensor (Biospherix). After hyperoxia the cell pellets were collected by centrifugation and pellets were suspended in lysis buffer (20 mM Tris HCL, pH7.4, 150 mM Nacl, 0.5% Triton-x 100) and supernatant was collected after centrifugation at high speed 14000 g for 15 minutes at 4 degrees Celsius. The amount of protein was calculated by BCA assay kit (Pierce, Rockford, Ill.). Equivalent amount of protein was (15μg) influenced to SDS-PAGE by 4-20% tris-glycine gel (Bio-rad) after that subjected to electro transfer with PVDF membrane followed by blocking in 5% skim milk with washes with TBST. The membranes are treated with primary and secondary antibodies. The bands were developed by pierce EC1 (Thermo Fischer scientific) and film with protein bands were scanned and analyzed densitometrically by NIH ImageJ software. The ratio of protein to loading control was taken and analyzed in percentages.

JC-1 staining

HMVEC-L cells were maintained in a EGM-2 (Lonza) media supplied with FBS (25 ml), Hydrocortisone (0.2 ml), hFGF-B (2.0 ml), VEGF (0.5 ml), R3 IGF-1 (0.5 ml), Ascorbic acid (0.5 ml), hEGF (0.5 ml), GA-1000 (0.5 ml) at 37 degrees Celsius in a 5% carbon dioxide humidified incubator to maintain a sufficient cell growth. The HMVEC-L cells were plated at density of 1×10000 cells in cell view dish with glass bottom (Thomas scientific). When cells attained 70% confluent, they were exposed to hyperoxia for 48 hours with Alda-1 and without Alda-1. Followed by hyperoxia the HMVEC-L cells were washed by HBSS solution (without ca+ and mg+) (Gibco). After washes the HMVEC-L was treated with JC-1 (5 μM) (Thermo Fisher) at 37 degrees for 15 minutes and cells were washed in Hbss for three times and placed in the medium. The live cell imaging was conducted in fluorescence microscopy (Olympus). The green and red images were captured in fluorescence and images were quantified by image J by red to green ratio.

ALDH2 activity

The enzymatic activity of ALDH2 was measured by transformation of acetaldehyde to acetic acid (Chen et al., 2008). The HMVEC-L cells were cultured as mentioned above, followed by 300 μl of buffer (10 mM DTT, 100 mM Tris-HCl pH 8.0, 20% glycerol, 1% Triton X-100) and centrifuged at 55000 g for 30 minutes at 4 degrees. The supernatant is utilized to detect activity of ALDH2 at 340 nm in a spectrophotometer. The assay mixture (1 ml) has 10mM NAD+, 100 mM sodium pyrophosphate, acetaldehyde, water, 100 μig mitochondrial lysate. The assay activity was begun by adding acetaldehyde (10 mM) to the cuvette. The specificity of enzyme reaction was expressed as nmol NADH/minute/mg protein.

Statistical Analysis

All experiments were conducted with 3 per group and values were indicated as means±SE. Statistical significance was calculated by using Microsoft excel and T-test were 2 tailed and values less than P<0.05 were considered as significant.

Results.

Aldh2 activator attenuates hyperoxia-induced 4HNE accumulation in pulmonary endothelial cells

Alda-1 has been shown to protect many different cell types against oxidative stress by increasing the ALDH2 activity. However, no study has revealed the similar protective effect of Alda-1 on lung endothelial cell. To verify its anti-oxidative effect on lung endothelial cells, we exposed the human microvascular endothelial cells (HMVEC) to hyperoxic conditions in the presence and absence of Alda-1. The results show that pretreatment with Alda-1 attenuates hyperoxia induce 4HNE increase in human microvascular endothelial cells. Hyperoxia causes 55% increase in 4HNE compared to Normoxia. pretreatment with Alda-1 in hyperoxic conditions causes 26% decrease compared to hyperoxia. These results indicate that aldh2 plays a vital role in protecting lung vascular endothelial cells from oxidative stress induced 4HNE upregulation, which is a trigger for cell apoptosis. FIG. 5 shows a graph that represent the whole cell protein lysate for Human microvascular endothelial cells under different conditions subjected to ELISA to evaluate the levels of 4HNE content during the Normoxia, hyperoxia, hyperoxia+Alda1 (20 μM) for 48 hours.

Aldh2 activator attenuates hyperoxia-induced downregulation of oxidative stress in pulmonary endothelial cells

To determine whether aldh2 activation suppress apoptosis signaling caused by oxidative stress in human microvascular endothelial cells, the protein levels of Bax and cytochrome C in whole cell lysate were quantified by western blotting. The results demonstrate that hyperoxia causes 2.5-fold significant increase and 1.5-fold increase in cytochrome C levels and BAX levels respectively compared to normoxia. The pretreatment of ALda-1 in hyperoxia caused 64% significant and 25% significant decrease in cytochrome C and Bax respectively compared to hyperoxia without Alda-1 pretreatment. These results indicate that ALDH2 activation helps to protect Human microvascular endothelial cells from oxidative stress induced apoptosis. FIG. 6A shows the western blot analysis of the whole cell lysate for human microvascular endothelial cells under different conditions (n=3) subjected evaluate the levels of cytochrome C during normoxia, hyperoxia and hyperoxia with Alda-1 (20 μM) for 48 hours. Equal amounts of protein loaded per lane (20 μg). FIG. 6B shows expression of Cytochrome C was normalized to Beta actin and presented in arbitrary units. The results are shown in mean±SEM.

ALDH2 activator promotes hyperoxia-induced expression of ALDH2 in pulmonary endothelial cells

In literature Alda-1 treatment has been shown to have no impact on the expression of ALDH2 expression in invitro in many different cell types. Moreover, no study has been conducted revealed the expression of ALDH2 in lung microvascular endothelial cells. To confirm the effect of Alda-1 on ALDH2 expression, the protein level of ALDH2 in whole cell lysate was evaluated by western blotting. The result suggests that pre-treatment with Alda-1 in hyperoxia has no effect on ALDH2 expression in hyperoxia treatment. These results indicate that pretreatment with ALDH2 activator does not alter the expression of ALDH2 during hyperoxia in human microvascular endothelial cells. FIG. 7A shows a graph that depicts the whole cell lysate for human microvascular endothelial cells under different conditions (n=3) subjected to Immuno blot to evaluate the levels of ALDH2 during normoxia, hyperoxia and hyperoxia with Alda-1 (20 μM) for 48 hours normalized against Beta actin. The values indicate standard error mean (*P<0.05). Equal amounts of protein loaded per lane (20 μg). FIG. 7B shows expression of ALDH2 was normalized to Beta actin and presented in arbitrary units. The results are shown in mean±SEM.

ALDH2 activator promotes hyperoxia induced ALDH2 activity in pulmonary endothelial cells

To evaluate whether ALDH2 activator enhances ALDH2 activity during hyperoxia in human microvascular endothelial cells, the mitochondrial extract was isolated and ALDH2 activity assay was conducted. The results demonstrated that hyperoxia with Alda-1 treatment caused 45% increase in ALDH2 expression compared to hyperoxia without Alda-1 pretreatment. These results indicate that ALDH2 activator increases the activity of ALDH2 under hyperoxia in human microvascular endothelial cells. FIG. 8A shows a graph that depicts the whole cell lysate for human microvascular endothelial cells under different conditions (n=3) subjected to Immuno blot to evaluate the levels of Bax during normoxia, hyperoxia and hyperoxia with Alda-1 (20 μM) for 48 hours normalized against Beta actin. The values indicate standard error mean (*P<0.05). Equal amounts of protein loaded per lane (20 μg). FIG. 8A shows expression of Bax was normalized to Beta actin and presented in arbitrary units. The results are shown in mean±SEM.

ALDH2 activator promotes the hyperoxia induced mitochondrial membrane potential in pulmonary endothelial cells

To evaluate the effect of Alda-1 during hyperoxia on human microvascular endothelial cells the mitochondrial membrane potential was quantified by JC-1. The JC-1 is considered as an indicator of cellular damage and cell death. The cellular damage can be attributed by decrease in fluorescence red to green ratio. The results demonstrate that membrane potential is 1.90 fold increase in normoxia compared to hyperoxia and 1.62-fold significant increase in hyperoxia with Alda treatment compared to hyperoxia. These results suggest that Aldh2 activator decreases the cellular damage during hyperoxia in human microvascular endothelial cells (P<0.005). FIG. 9A shows a graph that shows the human microvascular endothelial cells under different conditions subjected to the JC1 staining to evaluate the mitochondrial membrane potential during normoxia, hyperoxia and hyperoxia with Alda-1 (20 μM) for 48 hours. The values indicate standard error mean (*P<0.05). FIG. 9B depicts Jc1 images were quantified using imageJ software and an intensity was expressed in arbitrary units. The results are shown in mean±SEM (Magnification=200×).

Discussion. Hyperoxia exposure causes cellular dysfunction in HMVECs, and prolonged exposure to hyperoxia causes organ failure (7). During hyperoxic exposure, the cell undergoes oxidative stress due to an increase in ROS (21). Although Alda-1 has been shown to reduce injuries in the heart, liver, brain, kidney, and intestines, it has not been thoroughly tested in the lung (12, 22-25). Aldh2 has also been implicated in numerous pathologies, such as ischemia, Alzheimer's, and Parkinson disease. The use of Alda-1 has been proven to attenuate endothelial cell injuries in neural and umbilical cells. For the first time, we have demonstrated the use of Alda-1, an Aldh2 activator, attenuating endothelial dysfunction in pulmonary human microvascular endothelial cells (HMVECs) during hyperoxia. Mitochondrial Aldh2 plays a pivotal role in preventing the accumulation of 4HNE (15, 18), oxidative stress (8), expression of apoptotic marker, and enhancement in mitochondrial membrane (15, 26).

It has been effectively shown that in vitro exposure to hyperoxia causes an accumulation of the toxic compound 4HNE causes excessive alveolar protein leak, and pulmonary edema contributing to ALI (26). For the first time we provide evidence that 4HNE causes extensive damage in HMVEC'S. We pretreated HMVECs with Alda-1 and exposed them to 48 hours of hyperoxia and determined that hyperoxic exposure caused an accumulation of 4HNE, but that treatment with Alda-1 reduced the amount. Hyperoxic exposure in vivo has been shown to increase the formation of adducts of 4HNE (27). Moreover, these protein alterations result in impaired cellular feedback, thereby causing the production of ROS and oxidative stress, ultimately leading to cellular dysfunction and death (8, 16).

The exposure of pulmonary cells to hyperoxia for prolonged durations causes damage to epithelial and endothelial cell barriers, thereby increasing ROS, oxidative stress, and apoptosis, and decreasing the mitochondrial membrane potential (9, 28). Hyperoxia also causes an increase in 4HNE levels affecting mitochondrial membrane potential. These changes occur within 48 hours of exposing HMVECs to hyperoxia. The disaggregation and death of HMVEC can be seen in hyperoxia (FIG. 5). The increase in mitochondrial membrane potential, Aldh2 activity and Aldh2 expression of HMVECs pretreated with Alda-1 (FIGS. 9A, 10 and 7A respectively) shows mitochondrial oxidative stress induced by hyperoxia.

For the first time, we have revealed that, in HMVECs exposed to hyperoxia, Aldh2 activity and aldh2 expression increase as a result of the Alda-1 treatment during hyperoxia (FIGS. 7A and 10). When treated with Alda-1 prior to the hyperoxia exposure, we saw that the recovery of Aldh2 decreased cytochrome C release and repaired mitochondrial membrane potential (FIGS. 6A and 9A). Alda-1 provided a powerful protection against the effects of hyperoxia in HMVECs, as shown by the diminished levels of Bax, an apoptotic activator indicative of mitochondrial stress (FIG. 8A). The importance of Alda-1 shielding Aldh2 against these disastrous effects has been profoundly described by Perez Miller et al.

We demonstrate for the first time the protective effects of Alda-1 in HMVCEs undergoing 48 hours of hyperoxic exposure. The pretreatment of Alda-1 before hyperoxic exposure caused a decrease in 4HNE levels versus HMVECs that were exposed to hyperoxia alone (FIG. 5). This is because ALDH2, which has increased activity in cells treated with Alda-1, converts 4HNE into acetaldehyde, which is then converted into acetic acid by ALDH2 as well (Xu, Guthrie et al. 2006). There was a no change in the expression of ALDH2 in the group treated with DMSO or Alda-1 prior to hyperoxia exposure (FIG. 7). The hyperoxic ALDH2 expression levels were high when compared to normoxia because ALDH2 exerts protective effects during hyperoxic exposure, thereby leading to its natural overexpression. Overall, Alda-1 was successfully able to enhance the activity ALDH2 in hyperoxic conditions to relieve oxidative stress. Literature shows that ALDH2 is a critical protein in numerous tissues, such as liver, heart, muscle, and kidney (30), and for the first time we demonstrate its importance in lung tissues (Human Lung Microvascular Endothelial cells).

In summary, this disclosure suggest that hyperoxic damage can be guarded by ALDH2 activation via pretreatment with Alda-1. For the first time the effects of Alda-1 in human microvascular endothelial cells during hyperoxia have been shown. It has been shown that treatment with Alda-1 diminishes the accumulation of 4HNE and reduces oxidative stress by decreasing ROS during hyperoxia. Hyperoxia has been shown to elevate oxidative stress, depolarize the mitochondrial membrane, decrease ALDH2 activity, disrupt cellular transduction, and cause apoptosis. The use of Alda-1 reversed the harmful effects of hyperoxic injury to HMVECs, revealing that oxidative stress is important in the pathological processes of various diseases associated with the lung and endothelial cells. Therefore, administration of Alda-1 may act as a novel therapeutic drug to mitigate hyperoxic injury in lungs.

Example 4

Introduction. Acute respiratory distress syndrome (ARDS) is associated with fluid filled lungs and hypoxia. Thus, ARDS patients are placed on supplemental oxygen; however, hyperoxia can damage the lungs, cause mitochondrial dysfunction, and ultimately lead to acute lung injury in animal models of ARDS. Mitochondrial aldehyde dehydrogenase 2 (ALDH2) metabolizes dangerous, reactive products, such as 4-hydroxynonenal, that otherwise causes oxidative stress. This disclosure determines whether Alda-1, an ALDH2 activator enhances the activity of ALDH2, thus alleviating hyperoxia damage by preventing mitochondrial dysfunction and reducing immune cell infiltration. It also determined if administration of Alda-1 recovers mitochondrial dynamics and reduces cytokine levels and immune cell infiltration.

Methods. C57BL/6 mice were exposed to hyperoxia for 48 hours with DMSO (Control) and Alda-1 (20 mM) via alzet pumps on the dorsal side of mice. The mice were divided into four groups: normoxia+DMSO (room air), normoxia+Alda-1, hyperoxia+DMSO, and hyperoxia+Alda-1. The lungs were harvested and the bronchoalveolar lavage (BAL) fluid was collected and analyzed by immunoblot and cytokine levels.

Results. Immunoblot analysis of lung lysate demonstrates that mice treated with Alda-1, compared to mice treated with DMSO, during hyperoxia have decreased levels of OPA1 and Drpl, indicating that Alda-1 shields against oxidative stress for proteins associated with mitochondrial dynamics. Moreover, the BAL fluid analysis reveals less infiltration of macrophages in mice treated with Alda-1 versus mice treated with DMSO alone. These results indicate that activation of ALDH2 via Alda-1 is protective against hyperoxia-induced acute lung injury and may be a viable therapeutic agent for ARDS.

Conclusion. These findings suggest that Alda-1, an ALDH2 activator, maintains mitochondrial homeostasis and reduces immune cell infiltration in this mouse model of ARDS and may be a novel therapeutic agent in ARDS.

Example 5

Introduction. Acute Lung Injury (ALI) is characterized by acute and severe inflammation of the lungs that can result in respiratory failure. The main symptom of ALI is shortness of breath associated with low oxygen. It is characterized by X-ray findings of bilateral pulmonary infiltrates. The most common treatment for ALI is to provide supplemental oxygen, which can lead to the accumulation of reactive oxygen species (ROS) and the toxic metabolite 4-hydroxy-2-noneal (4HNE). This leads to further oxidative stress, thus limiting cell proliferation and triggering aberrations in mitochondrial dynamics. Mitochondrial dysfunction is one of the hallmarks of ALI. Mitochondrial Aldehyde dehydrogenase 2 (ALDH2) acts as a mitochondrial shield against damage. Activation of ALHD2 via Alda-1 also can attenuate the mitochondrial damage during hyperoxic exposure. This example is focused on the therapeutic potential of Alda-1 to reduce the effects of hyperoxic exposure in ALI by protecting mitochondrial dynamics.

Methods. C57BL/6 mice were pretreated with DMSO (control) and 20μM Alda-1 (administered via Alzet pumps), then exposed to hyperoxia for 48 hours. The mice were divided into three groups: Room air, hyperoxia (48 hour)+DMSO, and hyperoxia (48 hour)+Alda-1. The lung tissues were harvested and lysates evaluated by immunoblot analysis.

Results. Western blot analysis of lung lysates indicates that mice treated with Alda-1 during hyperoxia versus mice treated only with hyperoxia have decreased levels of mitochondrial fusion proteins mitofusin 1 (MFN1) and mitofusin 2 (MFN2) relative to mice that were not treated. In addition, mitophagy protein PTEN-induced kinase 1 (PINK1) levels also decrease in mice treated with Alda-1 prior to hyperoxia exposure compared to hyperoxic controls. These data suggest that Alda-1 protects mitochondria against hyperoxia-induced damage by maintaining mitochondrial homeostasis.

Conclusion. These findings suggest that Alda-1 may be a treatment option to regulate the mitochondrial dynamics via activation of the mitochondrial enzyme Aldehyde dehydrogenase 2 (ALDH2).

Example 6

Introduction. Acute lung injury (ALI) is a critical lung disorder where the inefficient oxygen uptake causes acute hypoxemia and Acute Respiratory Distress Syndrome (ARDS). There are nearly 200,000 annual cases of ALI in the United States alone, and the incidence rate is increasing. At the molecular level, hyperoxic exposure causes an increase in reactive oxygen species, leading to an accumulation of 4-Hydroxy-2-nonenal (4HNE), a lipid peroxidation product causes protein adducts and inhibits the activity of the mitochondrial enzyme ALDH2 (Aldehyde dehydrogenase), which is involved in metabolism of alcohol. Use of Alda-1 may reduce the effect of acute lung injury.

Methods. The C57BL/6 mice were embedded with osmotic pumps and continuously injected with either DMSO or Alda-1 (20 μM), then they were exposed to 100% oxygen 48 hours. The lung samples, as well as Bronchial Alveolar Lavage Fluid, were collected for the evaluation of infiltration of cytokines, inflammation, autophagy, and apoptosis by Diff Kwik staining, H&E staining, and western blot.

Results. The Mice treated with DMSO in hyperoxia showed more cytokine and neutrophil infiltration and elevated inflammation than mice treated with Alda-1 in hyperoxia. The western blot analysis of Alda-1-treated mice showed reduced cytochrome C release, reduced LC3B, and reduced NF-κB-p65, while also showing a decrease in oxidative stress, inflammation and autophagy.

Conclusion. The findings imply that Alda-1, an ALDH2 activator is a potential therapeutic drug for the treatment of acute lung injury.

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The advantages set forth above, and those made apparent from the foregoing description, are efficiently attained. While the disclosure is susceptible to various modifications and implementation in alternative forms, specific embodiments have been shown by way of non-limiting example in the drawings and have been described in detail herein. Since certain changes may be made in the above construction without departing from the scope of the instant application, it is intended that all matters contained in the foregoing description or shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense.

The disclosure is not intended to be limited to the particular forms disclosed. Rather, the disclosure is to cover all modifications, equivalents, and alternatives falling within the scope of the disclosure as defined by the following appended claims and their legal equivalents.

Without further elaboration, it is believed that one skilled in the art can use the preceding description to utilize the present disclosure to its fullest extent. The examples and embodiments disclosed herein are to be construed as merely illustrative and exemplary and not a limitation of the scope of the present disclosure in any way. It will be apparent to those having skill in the art, and having the benefit of this disclosure, that changes may be made to the details of the above-described embodiments without departing from the underlying principles of the disclosure herein.

It is also to be understood that the following claims are intended to cover all of the generic and specific features of the invention herein described, and all statements of the scope of the invention that, as a matter of language, might be said to fall therebetween. 

What is claimed is:
 1. A method of preventing endothelial cell injury from hyperoxic injury, the method comprising: administering a therapeutically effective amount of Alda-1; administering a pharmaceutically acceptable excipient; wherein the Alda-1 activates aldehyde dehydrogenase 2 (ALDH2); wherein the Alda-1 reduces damage to endothelial cells from oxidative stress.
 2. The method of claim 1, wherein the Alda-1 rescues mitochondrial membrane potential.
 3. The method of claim 1, wherein the Alda-1 decreases cytochrome C release, reduced LC3B, and reduced NF-κB-p65.
 4. The method of claim 1, wherein the Alda-1 suppresses mitochondrial reactive oxygen species (ROS) production in human microvascular endothelial cells (HMVECs) under physiological conditions.
 5. The method of claim 1, wherein the Alda-1 decreases mitochondrial fusion protein mitofusin 1 (MFN1), mitofusin 2 (MFN2), and mitophagy protein PTEN-induced kinase 1 (PINK1).
 6. A method of treating pulmonary disease characterized by endothelial dysfunction, the method comprising: administering a therapeutically effective amount of Alda-1 to a subject suffering from the pulmonary disease characterized by endothelial dysfunction; administering a pharmaceutically acceptable excipient; wherein the Alda-1 activates ALDH2; wherein the Alda-1 reduces damage to endothelial cells from oxidative stress.
 7. The method of claim 6, wherein the Alda-1 rescues mitochondrial membrane potential.
 8. The method of claim 6, wherein the Alda-1 decreases oxidative stress, inflammation and autophagy.
 9. The method of claim 6, wherein the Alda-1 suppresses mitochondrial reactive oxygen species (ROS) production in human microvascular endothelial cells (HMVECs).
 10. The method of claim 6, wherein mitochondria homeostasis is preserved.
 11. The method of claim 6, wherein a unit dosage form of Alda-1 is used in an amount sufficient to reduce damage to endothelial cells from oxidative stress.
 12. The method of claim 6, wherein the pulmonary disease is acute respiratory distress syndrome (ARDS).
 13. The method of claim 6, wherein the pulmonary disease is acute lung injury (ALI).
 14. The method of claim 6, wherein the Alda-1 decreases 4-hydroxy-2-noneal (4HNE) in endothelial cells. 